Sperm can be collected from a great variety of mammals and then separated into X-chromosome bearing and Y-chromosome bearing populations based upon the difference in DNA content. In some conventional methods of spermatozoa separation, the DNA content of the spermatozoa to be separated can be stained with a fluorochrome(s) that upon excitation emit(s) a measurable amount of fluorescence. Because X-chromosome bearing spermatozoa contain a greater amount of DNA than Y-chromosome bearing spermatozoa, each X-chromosome bearing spermatozoa has the capacity to bind a relatively greater amount of fluorochrome than the corresponding Y-chromosome bearing spermatozoa. Comparison of the relative magnitude of emitted fluorescence upon excitation of the fluorochrome(s) allows the isolation of X-chromosome bearing spermatozoa from Y-chromosome bearing spermatozoa as described by U.S. Pat. No. 5,135, 759, hereby incorporated by reference.
Even though X-chromosome bearing spermatozoa and Y-chromosome bearing spermatozoa have been differentiated by and separated based upon the difference in emitted fluorescence for many years, and even though there is large commercial market for isolated populations of X-chromosome bearing spermatozoa and Y-chromosome bearing spermatozoa, there remain significant problems yet to be resolved.
A significant problem with conventional methods of separating X-chromosome bearing spermatozoa from Y-chromosome bearing spermatozoa can be that each resulting population contains a significant number of incorrectly separated spermatozoa that belong in the other population. This problem in differentiating between spermatozoa can, in part, be attributed to the lack of uniformity in the amount of fluorochrome bound to the spermatozoal DNA. As such, a range in the amount of fluorochrome bound by X-chromosome bearing spermatozoa is generated and a range in the amount of fluorochrome bound by Y-chromosome bearing spermatozoa is generated. When these ranges in the amount of fluorochrome overlap or yield some values that are similar, it can be difficult or impossible to classify those individual spermatozoa to one population or the other with any degree of certainty and cross contamination of the populations can occur.
This particular problem can be exacerbated with regard to spermatozoa obtained from frozen and subsequently thawed mammalian semen. The mean purity for separated Y-chromosome bearing spermatozoa population derived from previously frozen-thawed semen can be 85% or less, and the mean purity for separated X-chromosome bearing spermatozoa population derived from previously frozen-thawed semen can be 82% or less.
Another significant problem associated with staining of spermatozoal DNA can be the detrimental effects on fertilization rates and subsequent embryonic development of fertilized oocyte(s) (oocyte, ootid, or ovum, or a plurality of same, as may be appropriate within a specific application). One aspect of this problem may be that the amount of stain bound to the DNA may effect the viability of the spermatozoa resulting in lower fertilization rates. Another aspect of this problem can be that the amount of time that elapses during the staining of the DNA may effect the viability of the sperm resulting in lower fertilization rates. Another aspect of this problem may be that the amount of time that elapses during staining of the DNA may lower subsequent cleavage rates of oocytes fertilized with such stained spermatozoa. A 20% decline in cleavage rates have been observed for oocytes when staining time requires 190 minutes as compared to when staining time requires 60 minutes. Another aspect of this problem may be that the percent of oocytes fertilized with stained spermatozoa that proceed to blastulation may be lower as described in the journal article entitled “In vitro Fertilization with Flow-Cytometrically-Sorted Bovine Sperm”, Theriogenology 52: 1393-1405 (1999), hereby incorporated by reference herein.
Another significant problem may be that cryopreserved sperm may demostrate increased capacitation, and the length of time such spermatozoa are viable may be shortened. As such, if previously frozen spermatozoa are to be separated into X-chromosome bearing and Y-chromosome bearing populations that are to be subsequently used in applications such as in-vitro fertilization, in-vivo artificial insemination, or the like, then routine staining procedures may have to be abbreviated to maintain suitable number of viable sperm cells.
As relating to the problems of staining spermatozoa uniformly, even when spermatozoa are obtained from previously frozen-thawed semen; maintaining sperm viability; separating stained spermatozoa into X-chromosome bearing and Y-chromosome bearing populations, even when the spermatozoa being separated are obtained from previously frozen semen; generating populations of X-chromosome bearing and Y-chromosome bearing spermatozoa having high purity; and successfully using separated spermatozoa for artificial insemination, surgical insemination, and in-vitro fertilization techniques it can be understood there are significant problems with conventional technology which are addressed by the instant invention.